CN114438040A

CN114438040A - Monoclonal cell strain secreting antibody with PEDV (porcine epidemic diarrhea Virus) neutralizing activity and application thereof

Info

Publication number: CN114438040A
Application number: CN202011216592.2A
Authority: CN
Inventors: 曹永长; 薛春宜; 柳玉芳
Original assignee: Sun Yat Sen University
Current assignee: Sun Yat Sen University
Priority date: 2020-11-04
Filing date: 2020-11-04
Publication date: 2022-05-06

Abstract

The invention discloses a monoclonal cell strain secreting an antibody with PEDV (porcine epidemic diarrhea Virus) neutralizing activity and application thereof, belonging to the technical field of biology. The name of the monoclonal cell strain is hybridoma cell strain 2B11, and the preservation number is CCTCC NO: c2020188, deposited at the China center for type culture Collection, located at the university of Wuhan, China, in 2020, 9, 26. It can be used for preparing antibodies with PEDV neutralizing activity. The invention also provides a method for detecting the titer of the PEDV vaccine, which has the advantages of good specificity, high sensitivity and simple and convenient operation, can quickly detect the level of PEDV neutralizing antibodies in immune pig serum in batches, can evaluate the immune effect of the PEDV vaccine, and fills the gap of the conventional method for quickly detecting the level of the PEDV antibodies.

Description

Monoclonal cell strain secreting antibody with PEDV (porcine epidemic diarrhea Virus) neutralizing activity and application thereof

Technical Field

The invention relates to a monoclonal cell strain secreting an antibody with PEDV (porcine epidemic diarrhea Virus) neutralizing activity and application thereof, belonging to the technical field of biology.

Background

PEDV is a pathogen of Porcine Epidemic Diarrhea (PED), is a single-stranded positive-strand RNA virus, and belongs to the order nidovirales, family coronaviridae, genus alphacoronavirus. This viral infection can lead to diarrhea in weaned piglets, dehydration in suckling piglets, thinning of the small intestinal wall of pigs, villous atrophy and immediate death in severe cases. The symptoms after infection with this virus are very similar to those of other coronavirus infections and therefore cannot be distinguished by clinical diagnosis. This makes the control of diarrhea in clinical production extremely difficult and at the same time presents a great challenge to the clinical use of vaccines. The disease can infect swinery of all ages, the morbidity and mortality of the suckling piglets are high, and especially the mortality of the suckling piglets within 7 days of age can reach 100 percent after infection.

Vaccines are the main means for controlling PED, but how to effectively assess antibody levels after immunization is a difficult problem that plagues the whole industry. ELISA has been many times proven to be an ideal tool for assessing antibody levels because of the advantage of being able to rapidly test samples in large quantities. At present, two ELISA antibody detection kits related to PEDV are available on the market, one is an anti-N protein antibody detection kit produced by BIOVET company of Canada, and can be used for evaluating the infection condition of the PEDV of the swinery. The other is a quantitative detection kit for IgA antibody in milk produced by Korea Anjie, which can be used for evaluating the IgA level in the milk of sows.

Antibodies are divided into neutralizing and non-neutralizing antibodies, and the relationship between neutralizing antibodies and virus protection has been demonstrated in many viral vaccine screening processes. IgA and anti-N protein antibody levels are not sufficient to assess the effectiveness of the vaccine and there is no theoretical basis for the correlation between the two and immune protection. Although the plaque reduction test is a gold standard for detecting the level of PEDV neutralizing antibodies, the method has the advantages of good specificity, high sensitivity and the like, but has the defects of long test period, requirement for operating live viruses and cells, high experimental environment requirement and the like, and is very unfavorable for large-scale application under simple conditions such as a pig farm and the like. Due to the specificity of coronaviruses, no neutralizing antibody ELISA test kit has been developed for detecting PEDV. Therefore, the establishment of a neutralizing antibody competition ELISA method is an ideal scheme for solving the difficulty in antibody monitoring and evaluation after PEDV immunization.

Disclosure of Invention

The primary object of the present invention is to overcome the drawbacks and disadvantages of the prior art and to provide a monoclonal cell line secreting antibodies having PEDV neutralizing activity.

Another objective of the invention is to provide an application of the monoclonal cell strain.

It is a further object of the present invention to provide applications of the above detection method.

The purpose of the invention is realized by the following technical scheme:

a monoclonal cell strain secreting antibodies with PEDV neutralizing activity is named as a hybridoma cell strain 2B11, and the preservation number is CCTCC NO: c2020188, deposited at the China center for type culture Collection, located at the university of Wuhan, China, in 2020, 9, 26.

The monoclonal cell strain secreting the antibody with the PEDV neutralizing activity is applied to preparation of the antibody with the PEDV neutralizing activity.

An antibody having PEDV-neutralizing activity, which is prepared from the monoclonal cell line.

The antibody with the PEDV neutralizing activity is applied to detection of the titer of the PEDV vaccine.

The PEDV vaccine titer refers to the level of neutralizing antibodies contained in serum of a pig immunized with the PEDV vaccine.

A method of detecting PEDV vaccine titer comprising the steps of:

(1) marking the antibody with PEDV neutralization activity by a chromogenic marker to obtain a marked antibody;

(2) the labeled antibody and the serum to be detected are reacted with PEDV together according to OD_450mmCalculating inhibition rates before and after blocking, and judging whether the serum to be detected is positive or negative;

(3) and calculating to obtain the titer of the PEDV vaccine according to the inhibition rate of the serum to be detected and the standard curve.

The chromogenic label in the step (1) is preferably horseradish peroxidase (HRP).

The labeling in step (1) is preferably carried out by using a chromogenic labeling substance labeling kit.

The chromogenic marker labeling kit is preferably a horseradish peroxidase antibody labeling kit.

The step (2) preferably specifically comprises the following steps:

1) coating antigen: coating the enzyme label plate with the antigen PEDV-coated complete virus, sealing, and cleaning to obtain an enzyme label plate containing PEDV;

2) adding the serum to be detected and the labeled antibody into an ELISA plate containing PEDV for reaction;

3) after the reaction is finished, cleaning; adding color developing agent, developing in dark place, adding stop solution, and measuring OD_450mm；

4) According to OD_450mmAnd calculating inhibition rates before and after blocking, and judging whether the serum to be detected is positive or negative.

The coating in the step 1) comprises the following specific steps: the solution containing PEDV was added to the microplate, coated overnight at 4 ℃ and washed with PBST.

The solution containing PEDV is preferably a solution obtained by diluting a concentrated solution of PEDV with a coating solution.

The coating solution is carbonate buffer solution with pH of 9.6 and 0.05 mol/L.

The number of washing is preferably 3.

The blocking described in step 1) is preferably performed for 2h using a 3% (w/v) BSA solution.

The washing described in step 1) is preferably 3 times using PBST.

The coating concentration of PEDV in step 1) is preferably 20. mu.g/mL.

The concentration of the labeled antibody in step 2) is preferably 0.2. mu.g/mL.

The amounts of PEDV and labeled antibody added were 100. mu.L each.

The addition mode in the step 2) is preferably as follows: mixing the serum to be detected and the labeled antibody, and then adding an ELISA plate containing PEDV; more preferably: and diluting the serum to be detected by a PBS (phosphate buffer solution) in a multiple ratio, mixing the obtained diluent with the labeled antibody in the same volume, and adding the mixture into an ELISA plate containing PEDV.

The reaction in step 2) is preferably incubated at 37 ℃ for 1 h.

The washing described in step 3) is preferably washing with PBST.

The color developing agent in the step 3) is preferably TMB color developing.

The color developing agent in the step 3) is preferably prepared by the following steps: dissolving TMB dry powder in DMSO to obtain TMB mother solution, and storing at-20 deg.C in dark place; na with the concentration of 0.2mol/L is added before use₃HPO₄Uniformly mixing the solution and citric acid solution with the concentration of 0.1mol/L according to the volume ratio, and adding TMB mother solution and H₂O₂Obtaining a TMB working solution (i.e. a color developing agent) by dissolving, wherein the concentration of TMB is 0.1mg/mL, H₂O₂Has a concentration of 9X 10^-3％(v/v)。

The concentration of the TMB mother liquor is preferably 1 mg/mL.

The stop solution in the step 3) is preferably H₂SO₄(ii) a More preferably H at a concentration of 2mol/mL₂SO₄。

The inhibition rate in the step 4) is calculated according to the following formula: inhibition rate (1-OD)₄₅₀OD value of sample to be tested/OD value of blank control) 100%.

The judgment criteria in step 4) are preferably as follows: average value of negative serum inhibition rate

18.6, Standard Deviation (SD) 2.28; get

Negative cutoff, 21.9;

positive cutoff, 23.6; and if the secondary result is still suspicious, determining that the result is positive.

The standard curve in the step (3) is a curve related to the neutralization titer and the inhibition rate obtained by performing the reaction according to the serum with known titer and the labeled antibody in the step (2); the following are preferred: y is 14.48X +17.626, X is the neutralization potency, and Y is the inhibition rate.

Compared with the prior art, the invention has the following advantages and effects: the invention establishes a method for detecting the level of neutralizing antibodies of the Porcine Epidemic Diarrhea Viruses (PEDV) of the swinery after the swinery is immunized, and particularly, the method has the advantages of good specificity, high sensitivity and simple and convenient operation, can quickly detect the level of the PEDV neutralizing antibodies in the serum of immunized pigs in batches, can evaluate the immunization effect of PEDV vaccines, and supplements the vacancy of the existing method for quickly detecting the level of the PEDV antibodies.

Drawings

FIG. 1 is a graph showing the results of the identification of the purified PEDV virions; wherein A is a PEDV sucrose density gradient ultracentrifugation photo, B is an SDS-PAG detection result, and C is a Western blot detection result; in lanes B and C, protein Marker, lane 1, sucrose density gradient centrifugation 40% -60% and lane 2, sucrose density gradient centrifugation 20% -40%.

FIG. 2 is a graph showing the results of the sensitivity test of cELISA.

FIG. 3 is a graph of the results of the correlation of cELISA with neutralization assays.

Detailed Description

The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.

The PEDV positive serum is presented by doctor Zhou Qingfeng of research institute of Wen's food group Limited; PEDV GDS01 was isolated and deposited by the laboratory (PEDV GDS01 is disclosed in the references "neutral anti-bodies against genetic epidemic viral block virus attachment and interaction. Gong et al, virology Journal (2018)15: 133"); SP2/0 cells were obtained from Wuhan Virus institute, 37154, a comet researcher (SP2/0 cells were purchased via CCTCC). SPF-grade BABL/c mice were purchased from the Experimental animals center of Zhongshan university; vero cells are subcultured in the laboratory (the Vero cells can be purchased through CCTCC); EDTA-free pancreatin was purchased from Invitrogen; DMEM medium, pancreatin, fetal bovine serum were purchased from GIBCO; low melting point agarose was purchased from TaKaRa corporation; EZ-Link^TM Maleimide Activated Horseradish Peroxidase Kit was purchased from Thermo.

Example 1

(1) And (3) PEDV whole virus propagation:

after a cell culture flask t175 is laid in advance and the Vero cells of the monolayer are fully grown, the Vero cells are washed three times by PBS (pH7.4 and 0.01mol/L), and then 19mL of DMEM medium without serum and 1mL of PEDV GDS01 virus solution (the virus titer is 10) are added⁷TCID₅₀mL) and 200. mu.g pancreatin without EDTA, to allow the virus to adsorb into the cells, and incubating in an incubator at 37 ℃ for 1 h; discarding the supernatant, adding 20mL of serum-free DMEM medium and 200 μ g of EDTA-free pancreatin, continuously incubating in an incubator at 37 ℃ for 1-2d until the lesion reaches 90%, collecting the maintenance liquid and the lesion cells by using a centrifuge tube, and freeze-thawing for 2-3 times;

(2) concentration of PEDV whole virus:

a large amount of cultured PEDV GDS01 virus solution was centrifuged at 10000rpm and 4 ℃ for 30min to remove cell debris. In the experiment, polyethylene glycol (PEG 6000) is used for concentrating the virus, and the virus is purified by sucrose density gradient ultracentrifugation. The specific operation is as follows: adding 6-7% (w/v) PEG 6000 into the virus solution without cell debris, and standing overnight at 4 ℃; centrifugation was carried out at 10000rpm and 4 ℃ for 1 hour, followed by elution with a small amount of PBS. Three discontinuous sucrose concentration gradients of 20%, 40% and 60% are prepared, and virus concentrated solution is added to the uppermost layer for centrifugation. The mixture was centrifuged at 40000rpm at 4 ℃ for 2 hours, and the bands were collected. The bands of each layer were diluted with PBS and virus was ultrafiltered using a 30KD ultrafilter tube to wash out sucrose. PEG concentration and ultrafiltration of the ultrafiltration tube effectively reduce the times of virus super-separation and avoid virus spike protein falling off caused by overlong super-separation time. Ensuring the integrity of coronavirus particles is beneficial to the successful implementation of an immune experiment. The purification effect of the virus particles is identified by SDS-PAGE and Western blot of each layer of samples, and the result is shown in figure 1, which shows that the virus is concentrated and the virus protein is intact after purification.

(3) Preparation of PEDV monoclonal antibody

The purified virus is inactivated by 0.5% (v/v) beta-propiolactone, emulsified by Freund's adjuvant and then used for immunizing BALB/c female mice of 6-8 weeks old, and the specific immune route, immune dose and adjuvant selection are shown in Table 1. After four immunizations, mouse spleen cells were taken and fused with SP2/0 cells.Feeder cells were prepared prior to fusion as follows: female SPF BALB/c mice of 8 weeks old are taken, the eyeballs are picked and bled, and the serum is negative serum. The cervical vertebra is removed, and the cervical vertebra is soaked in 75% alcohol for 10 min. The abdominal skin is cut off in a clean bench to expose the peritoneum, 3mL of DMEM medium containing 20% fetal calf serum is sucked by an injector and injected into the abdominal cavity, the DMEM medium is sucked in situ and placed in a 50mL centrifuge tube, and the fresh medium is taken to repeatedly operate once to obtain the required macrophage. The peritoneum was cut open and the spleen aseptically removed and placed in a homogenizer. Adding 3mL of culture medium containing 20% fetal calf serum into a homogenizer, grinding and separating splenocytes, adding 3mL of culture medium containing 20% fetal calf serum, uniformly mixing, standing for 5min, sucking 3mL of supernatant into a 50mL centrifuge tube by a pipette, continuously adding 3mL of culture medium containing 20% fetal calf serum into the homogenizer, grinding, uniformly mixing, standing, sucking 3mL of supernatant, and repeatedly washing for 2 times. The collected supernatant was put into a macrophage tube. Centrifuging at 1000rpm for 10min to remove supernatant, resuspending in PBS, counting cells, diluting to 10⁵The cells were plated in 96-well plates at 100. mu.L/well and incubated overnight in a cell incubator.

The BALB/c mice after the boosting immunization are taken, the eyeball is picked for bleeding (positive serum), and then cervical dislocation is carried out for death. The skin of the lower abdomen of the mouse was grasped with forceps and the peritoneum was cut open to expose the spleen. The spleen was grasped with forceps and placed in a sterile homogenizer. Adding 3mL of culture medium containing 20% fetal calf serum into a homogenizer, grinding splenocytes, adding 3mL of culture medium containing 20% fetal calf serum, standing for 5min, sucking 3mL of supernatant containing 20% fetal calf serum into a 50mL centrifuge tube, continuously adding 3mL of culture medium containing 20% fetal calf serum into the homogenizer, standing for 5min after uniformly mixing, sucking 3mL of supernatant again, and repeating the washing for 2 times. The supernatant was removed by centrifugation at 1000rpm for 10min, and the cells were resuspended in PBS and counted. The SP2/0 cells and splenocytes were mixed in a cell number of 1:5 proportion, mixing evenly, centrifuging at 1500rpm for 10 min. The supernatant was discarded, the tip of the finger tapped at the bottom of the tube, and after the pellet was loosened slightly, the centrifuge tube was placed in a 37 ℃ water bath to maintain the temperature, 1mL of pre-warmed 50% (w/v) PEG was slowly added thereto, and the mixture was stirred slightly with a tip while being added, which had to be completed within 1 min. Then 10mL of pre-warmed medium was added at constant rate over 3 min. Centrifuging at 1000rpm for 5min, removing supernatant, and standing at 37 deg.C for 8 min. Suspending with HAT medium, and culturing in 96-well culture mediumIn the plate, 250. mu.L/well. 37 ℃ and 5% CO₂Culturing in an incubator.

After 10 days of fusion, the cell supernatants were slightly yellow. 100. mu.L of cell supernatant was taken per well as monoclonal antibody to be screened, and detected by indirect immunofluorescence. The method comprises the following specific steps: the Vero cells are passaged into a 96-well plate, and the virus is inoculated in an amount of 100TCID50/mL per well when the cells grow to 90% -100% fusion degree; when the cells are obviously diseased (about 24-36h), discarding the supernatant, washing with PBS for 3 times, and fixing with 4% paraformaldehyde for 15 min; discarding the supernatant, washing with PBS for 3 times, adding 100 μ L Triton X-100 with concentration of 0.5% (v/v) for cell permeation for 15 min; discarding the supernatant, washing with PBS for 3 times, adding 3% BSA, and blocking at 37 deg.C for 1 h; discarding the supernatant, washing with PBS for 3 times, adding 100 μ L of the cell supernatant to be screened, and incubating at 37 deg.C for 1 h; the supernatant was discarded, washed 3 times with PBS, and 100. mu.L of PBS was added in a volume ratio of 1:500 diluted secondary antibody (the secondary antibody is Cy3 labeled goat anti-mouse IgG antibody from Proteitech company) and incubated for 1h at 37 ℃; discard the supernatant, wash with PBS 3 times, observe with a fluorescence microscope, and take a photograph.

10 PEDV monoclonal antibodies were screened by this method. Positive hybridoma cells were diluted to less than 100 cells per plate and plated into 96-well plates to screen for monoclonal hybridoma cells. And injecting the monoclonal hybridoma cells into abdominal cavity of female mice for 10-12 weeks to prepare ascites, extracting ascites from the mice with obvious abdominal distension and action retardation after about 10 days, centrifuging at 12000rpm for 10min, collecting supernatant, and freezing at-80 ℃ for storage.

Table 1 mouse immunization procedure

(4) Protein G column purification of monoclonal antibodies

According to the procedure of the Protein G Sepharose 4Fast Flow protocol, the column was soaked in 20% (v/v) ethanol solution and 3 times the column volume ddH₂O, 3 column volumes in Binding Buffer (Binding Buffer). 2mL of ascites fluid was diluted to 10mL with a pre-cooled binding buffer, and impurities such as fat were filtered off with a 0.45 μm filter. Slowly adding the sample to the purification column while collecting the purificationAnd the subsequent effluent is discharged. Washing with binding Buffer 5 times the column volume, eluting with pre-cooled eluent (Elute Buffer)10mL, collecting the eluate with a 15mL centrifuge tube containing 1mL Tris-HCl solution (pH9.0, 1mol/mL), and adding 2mL Tris-HCl dropwise while collecting the eluate to prevent antibody denaturation and inactivation. And after the purification is finished, taking a small amount of antibody for detecting the purification effect of the antibody through electrophoresis, and quantifying the BCA after the purity is qualified. Diluting to a certain concentration, and then mixing the components according to the proportion of 1:100 adding protease inhibitor, packaging, labeling, and freezing for storage.

(5) Determination of neutralizing Activity and potency of monoclonal antibodies

PEDV GDS01 virus was first diluted to 200 PFU/mL with carbonate buffer (pH9.6, 0.05 mol/L). After the monoclonal antibody is diluted in a multiple proportion, the volume ratio of each gradient diluted antibody to the virus solution is 1:1 mixing (1 mL in total), incubating at 37 ℃ for 1h, inoculating to a 6-well plate full of a single-layer Vero cell, incubating at 37 ℃ for 1h, washing off the virus which is not specifically bound by PBS, adding a virus growth culture medium containing 1% low-melting-point agarose, standing for 20min, performing inverted culture in a cell incubator at 37 ℃ after the virus is solidified, staining with 0.03% neutral red for 30min after 36h, counting plaques, and taking the highest dilution of the antibody which does not produce the plaques as the neutralization titer of the monoclonal antibody. According to international standards, two neutralizing antibodies 2B11 (2.5. mu.g/mL < NC50< 5. mu.g/mL), 2G8 (5. mu.g/mL < NC50< 10. mu.g/mL) with high binding activity were successfully screened by this method. The monoclonal cell strain secreting the 2B11 antibody is named as a hybridoma cell strain 2B11, and the preservation number is CCTCC NO: c2020188, deposited at the China center for type culture Collection, located at the university of Wuhan, China, in 2020, 9, 26.

(6) Horse radish peroxidase labeled monoclonal neutralizing antibody

According to EZ-Link^TMThe steps of Maleimide Activated Horseradish Peroxidase Kit specification, 10mL of coupling buffer solution is taken and 90mL of PBS is added to prepare MCB. Add 100. mu.L MCB to 6mg of 2-MEA. 5mg of purified 2B11 antibody was added to a 2-MEA vial and incubated at 37 ℃ for 90 min. Using 30mL MCB equilibrium column, cooling the mixture to room temperature, loading on the column, adding MCB for elution, recovering 0.5mL antibody from 6 th to 10 th column with high efficiency, detecting and recovering NanodropAnd (4) concentration. The recovered antibody is added with activated HRP (activated HRP) and incubated for 1-2h at room temperature. Dialyzed overnight, diluted to 5mL, sampled for detection, the remainder was added with 50% (v/v) glycerol and 0.03% (w/v) sodium azide, and BCA quantitated and brought to-80 ℃ for use.

(7) Enzyme-labeled monoclonal antibody quality identification

0.3mL of HRP-2B11 without glycerol is diluted by 10 times, and a full-wavelength plate reader detects the absorbance at the wavelengths of 280nm and 403nm, and the amount of HRP-2B11 enzyme conjugate is determined by substituting the formula. Enzyme binding amount (mg/mL) ═ A₄₀₃X 0.40 x dilution factor. HRP 2B11 content (mg/mL) ═ A₂₈₀-A₄₀₃0.3). times.0.62 times the dilution factor.

Substituting into formula to calculate coupling efficiency:

the HRP content was 0.213 × 0.4 × 10 — 0.852 mg/mL.

The content of HRP-2B11 (0.194-0.213 0.3) 0.62-10 (0.806 mg/mL).

The ratio of total amount of labeled antibody to total antibody was 0.806mg/mL 5mL/5mg 100% 80.6%.

(8) ELISA for determining optimal coating amount of antigen and using concentration of enzyme-labeled antibody

And (3) largely propagating PEDV GDS01 according to steps (1) and (2), concentrating the virus by 100 times, purifying by a sucrose gradient to obtain a coating antigen, and determining the total protein amount by a BCA method. Each sample to be tested was repeated 3 times using a checkerboard method. The antigen was diluted in coating solution (pH9.6, 0.05mol/L carbonate buffer) at a double rate and the initial dilution concentration was 80. mu.g/mL. Add 96-well microplate longitudinally, 100. mu.L per well, overnight at 4 ℃. The supernatant was discarded the next day and washed 3 times with 200. mu.L PBST. mu.L of 3% (w/v) BSA, blocked for 2h at room temperature, washed 3 times with PBST. Adding 1: dilution of positive serum (neutralization titer 1:32, No. 3, by plaque reduction assay) and negative serum (No. 33), 100 μ L per well, incubation for 1h at room temperature, PBST washing 3 times. Adding into the mixture at a volume ratio of 1:500, 1:1000, 1:2000, 1:4000, 1:8000, diluting with HRP-2B11 (100 μ L per well), and incubating at 37 deg.C for 1 h. Wash 3 times, 100. mu.L of TMB color, 50. mu. L H₂SO₄(concentration: 2mol/L) to terminate the reaction, and measuring an OD450nm value by using a microplate reader. Selection of Positive serum inhibition/negative serum inhibitionThe maximum rate is the optimal coating amount and the working concentration of the enzyme-labeled antibody. The developer is prepared by dissolving 30mg of TMB dry powder (purchased from manufacturer) in 30mL of DMSO (purchased from Thermo company) and storing at-20 ℃ in the dark; 4.5mL of 0.2mol/L Na was added before use₃HPO₄Mixing with 4.5mL of 0.1mol/L citric acid, adding 1mL of TMB solution and 3 μ L of 30% (v/v) H₂O₂。

The sensitivity and accuracy of the method can be influenced by the corresponding antigen coating concentration and the working concentration of the enzyme-labeled antibody in ELISA, and a certain correlation exists between the antigen coating concentration and the working concentration. As shown in Table 2, when the concentrations of the envelope antigen and the enzyme-labeled antibody were specified, the ratio of the positive serum to the negative serum was maximized, which is the condition of the optimal reaction concentration of both. The inhibition rate calculation formula is as follows: inhibition ratio of (1-OD)₄₅₀OD value of sample to be tested/OD value of blank control) 100%. According to the test results, when the coating concentration is 20 mug/mL and the enzyme-labeled HRP-2B11 (the initial concentration is 0.806mg/mL), the concentration is diluted by 1: the difference in P/N was greatest at 4000, which is the optimal dilution for both.

TABLE 2 determination of optimal coating concentration and dilution of HRP-2B11 (%)

(9) Determination of serum dilution

According to the working concentration of the enzyme-labeled antibody determined above, setting the two serum samples to be 1: 1. 1: 2. 1: 4. 1:8 sample dilution tests (3 replicates are set), and the dilution at which the positive serum inhibition rate/negative serum inhibition rate ratio is maximum is the optimal dilution of the serum sample.

Taking PEDV positive serum and negative serum, and mixing the obtained mixture according to the volume ratio of 1: 1. 1: 2. 1: 4. 1:8, detecting the dilution of the sample, and when the dilution of the serum sample is 1:2, the P/N difference was greatest, the optimal serum dilution (Table 3).

TABLE 3 determination of optimal coating concentration and dilution of sample (%)

(11) Detection of competitive ELISA detection method for PEDV antibodies

1) Determination of cELISA Positive and negative judgment criteria

10 PEDV-negative sera (negative sera from samples taken without vaccination and determined to be negative by RT-PCR and IFA) were assayed for OD by established methods_450nmConversion of value, substitution formula, into inhibition ratio

Each sample was measured 3 times, Standard Deviation (SD) was calculated, and then relevant standards were established for judgment. Set according to statistical principles

And

and respectively determining the critical values of negative and positive, and performing secondary detection if the critical values between the negative and positive are suspicious, and determining the result as positive if the secondary result is still suspicious. And taking 10 positive sera to draw a positive value distribution map. Positive sera were analyzed for the setting of the cut-off value and the results are shown in table 4. Average value of negative serum inhibition rate

It was 18.6 and had a Standard Deviation (SD) of 2.28. Get

The negative cutoff value was 21.9.

Positive cut-off was 23.6. And if the secondary result is still suspicious, determining that the result is positive. The positive samples have high determination results and positive set values, and meet the requirements.

TABLE 4 results of 10 negative samples tested by cELISA

2) Sensitivity of the composition

Taking the neutralization titer 1: 64, in PBS, to give a 1: 32. 1: 16. 1: 8. 1: 4. 1: 2; 50 μ L of each diluted serum was mixed with an equal volume of HRP-2B11 (1: 4000); PBST washing whole virus coated overnight ELISA plate 3 times, each hole adding 100 u L mixed solution, 37 degrees C were incubated for 1 h; discard the supernatant, PBST wash 3 times, TMB color development, H₂SO₄Stopping the reaction, OD_450nmAnd (4) reading the value. The EXCEL software establishes absorbance and titer standard curves. See fig. 2. The curves are linear, with neutralizing titers in the antibody at 1:2 or more, the linear regression equation is-4.8951X +6.144, and the coefficient R is determined²＝0.9878。

3) Specificity of

6 kinds of positive serum (virus infection) with clear background sources, such as TGEV, SIV, PDCoV, PEAV, PrV, PRRSV and the like, are detected according to the established reaction conditions, and the inhibition rates are all lower than 21.9 percent (table 5) after conversion, so that the cross reactivity between the positive serum and the inhibition rates is proved, and the detection specificity of the enzyme-labeled antibody is good.

Table 5 c elisa assay for different serum cross-reactivities (n ═ 5)

4) Repeatability of

Intra-batch repetition factor: taking 3 samples, using enzyme-linked immunosorbent assay plates of the same batch, continuously detecting for 5 days under the same condition, 1 time per day, and calculating the intra-batch variation coefficient;

batch-to-batch repetition factor: taking 3 samples, using enzyme-labeled plates of different batches, continuously detecting for 5 days under the same condition, 1 time per day, and calculating the inter-batch variation coefficient;

coefficient of variation CV (coefficient of variance) Standard Deviation (SD)/mean

The results show that the intra-and inter-batch coefficient of variation is less than 10% (table 6), indicating that the method is stable and reproducible.

TABLE 6 c-analysis of the coefficient of variation between plates in the sample plate measured by ELISA

(12) Preliminary application of PEDV antibody competition ELISA detection method

(1) Detecting antibody titers in serum samples

And (3) taking 5 parts of negative and positive serum and PEDV, incubating for 1h at 37 ℃, performing cELISA detection, and judging the blocking effect according to the change conditions of the inhibition rate of the negative and positive serum before and after reaction. The results are shown in table 7, and the positive sera after virus blocking showed a significant decrease in inhibition rate by cfisa.

TABLE 7 blocking test results

PEDV positive serum (30 parts) and negative serum (10 parts) with clear background sources and different neutralization titers (plaque-determined titers) were examined using established reaction conditions, and the relationship between the inhibition rate and the neutralization titer was determined based thereon. The linear equation between the two is shown in fig. 3, and the result is that Y is 14.48X +17.626(X is the neutralization titer, and Y is the inhibition ratio), and the degree of fitting is 0.9591.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Claims

1. A monoclonal cell line that secretes antibodies having PEDV neutralizing activity, comprising: the name of the monoclonal cell strain is hybridoma cell strain 2B11, and the preservation number is CCTCC NO: c2020188, deposited at the China center for type culture Collection, located at the university of Wuhan, China, in 2020, 9, 26.

2. Use of the monoclonal cell line secreting antibodies having PEDV neutralizing activity according to claim 1 for the preparation of antibodies having PEDV neutralizing activity.

3. An antibody having PEDV neutralizing activity, comprising: prepared from the monoclonal cell strain of claim 1.

4. Use of an antibody having PEDV neutralizing activity according to claim 3 for detecting PEDV vaccine titer.

5. A method for detecting the titer of a PEDV vaccine, comprising the steps of:

6. The method for detecting the titer of PEDV vaccines according to claim 5, wherein:

the chromogenic marker in the step (1) is horseradish peroxidase;

the label in the step (1) is operated by a horseradish peroxidase antibody labeling kit.

7. The method for detecting the titer of PEDV vaccines according to claim 5, wherein:

the step (2) specifically comprises the following steps:

8. The method for detecting the titer of PEDV vaccines according to claim 7, wherein:

the specific steps of coating in step 1) are as follows: adding a solution containing PEDV into an enzyme label plate, coating overnight at 4 ℃, and washing with PBST;

blocking in the step 1) is performed by using 3% (w/v) BSA solution for 2 h;

the cleaning in the step 1) is PBST cleaning for 3 times;

the coating concentration of the PEDV in the step 1) is 20 mu g/mL;

the concentration of the labeled antibody in the step 2) is 0.2 mug/mL;

the adding amount of the PEDV and the labeled antibody is 100 mu L respectively;

the adding mode in the step 2) is as follows: mixing the serum to be detected and the labeled antibody, and then adding an enzyme label plate containing PEDV;

the reaction in the step 2) is incubated for 1h at 37 ℃;

the cleaning in the step 3) is PBST cleaning;

the color developing agent in the step 3) is TMB for color development;

the stop solution in the step 3) is H₂SO₄；

The inhibition rate in the step 4) is calculated according to the following formula: inhibition rate (1-OD)₄₅₀OD value of sample to be measured/OD value of blank control) 100%;

the judgment standard in the step 4) is as follows: yin (kidney)Mean value of sexual serum inhibition

18.6, Standard Deviation (SD) 2.28; get

Negative cutoff, 21.9;

9. The method for detecting the titer of PEDV vaccines according to claim 8, wherein:

the solution containing PEDV is obtained by diluting PEDV concentrated solution with coating solution;

10. The method for detecting the titer of PEDV vaccines according to claim 5, wherein:

the standard curve in step (3) is as follows: y is 14.48X +17.626, X is the neutralization potency, and Y is the inhibition rate.